Effects of structural rearrangements on the rheology of rennet-induced casein particle gels

During ageing of casein or skim milk gels, structural changes take place that affect gel parameters, such as pore size and storage modulus. These changes can be explained in terms of rearrangements of the gel network at various length scales. In this paper, rheological experiments on rennet-induced casein gels and a general model on rearrangements are presented. The results of experiments (e.g. microscopy, permeametry) and computer simulations, the model, and recent literature on casein gels and other types of particle gels are compared to each other. Experiments presented include measurements of storage and loss moduli and maximum linear strain of the casein gels. Parameters varied were pH (5.3 and 6.65) and temperature (25 and 30 degrees C). In addition, the casein volume fraction (5-9 vol.%) was varied, which enables application of fractal scaling models. For rennet-induced casein gels, it is demonstrated that at the lower pH, all types of rearrangements proceed significantly faster. The rearrangements include: an increase in the size of compact building blocks; partial disappearance of fractal structure; and the formation of straightened strands, some of which eventually break. All of these rearrangements seem to be a consequence of particle fusion. There are indications of universality of the relation between particle fusion and gel syneresis for gels composed of viscoelastic particles.     

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels.Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels.By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of κ-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk.This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.     

Closure characteristics of a thermally responsive single ion-track pore determined by size exclusion method

Cross-linked poly(N-isopropylacrylamide) (NIPAAm) gel was grafted on the wall of a single ion-track pore in poly(ethylene terephthalate) (PET). The opening and closing of the pore is controlled by temperature and observed by electric conductivity. In the shrunken state of the gel, ions and molecules can penetrate the membrane through the free volume of the pore. In the swollen state, the gel clogs the pore. Using mixtures of polyethylene glycol (PEG) of various molecular weights and 0.1 N potassium chloride, it was demonstrated that the responsive pore acts as a thermally controllable valve preventing the passage of PEG molecules larger than 2 nm. The mean value of the hydrogel mesh size is estimated to be (1.3±0.05) nm.     

Preparation and Characterization of Porous Titania by Modified Sol-Gel Method

Mesoporous titania, especially anatase, is attractive due to its potential applications. A novel method to control pore structure of titania, surfactant- or polymer modification, is proposed. The wet gels and gel films, prepared from Ti(O-nC₄H₉)₄ were dried at 90°C and annealed at 500°C after immersion in surfactant or polymer solutions, and mesoporous anatase was obtained. The pore size, pore volume and specific surface area of the surfactant-modified bulk gels, estimated from N₂ absorption-desorption curves, are more than twice larger than those of the gels without modification. The pore size of the surfactant-modified gel films, observed by SEM, are similar to that of the bulk gels. The pore size obviously depended on the size of micelles. The pore size of the gels modified with hydrophilic polymers hardly increased, but the pore volume and the specific surface area increased.     

Effect of size, proteic composition, and heat treatment on the colloidal stability of proteolyzed bovine casein micelles

The casein micelles of reconstituted nonfat milk that have been fractionated by controlled pore glass chromatography showed a relationship between their size and their proteic composition: The fractions containing the smaller particles were richer in κ-casein than the fractions containing the bigger ones, in accordance with the casein micelle model of submicelles. The initial aggregation rate of micelles of different sizes, partially proteolyzed with chymosin (para-casein micelles), was measured in conditions of enzyme excess in which aggregation is the rate-limiting step of enzymatic coagulation, showing higher rates for the smaller micelles with the production of less compact para-casein micelle networks. This behavior could be explained in terms of electrostatic and steric colloidal stabilization due to their lower negative net charge and size and to a higher surface density of hydrophobic “patches” of proteolyzed κ-casein related to a higher probability of effective collisions between particles. Differences in the β-casein content did not seem to affect the initial aggregation rate of the micelles. On the contrary, the modifications of the micelle surface by heating affected the colloidal stability of the hydrolyzed micelles in different ways. The denaturation of the whey proteins and the formation of covalent complexes with κ-casein modify the micelle surface, increasing specially the steric stabilization, and produces a diminution in the number of hydrophobic sites that could be able to give interparticle hydrophobic interactions.     

Yeast cells long-term interaction with asbestos fibers

Saccharomyces cerevisiae was supported on chrysotile, crocidolite and lixiviated chrysotile. Samples of the supported cells and free cells were observed by confocal laser scanning microscopy. After 30 days, the free cells showed no viability when stored at 30 °C, and a viability of 40% when stored at 4 °C. Supported cells stored at 30 °C were more viable than the free cells at early times, but showed no viability after 30 days. Samples stored at 4 °C showed that the adhered cells are more viable than the free cells, up to 30 days. Cells supported on chrysotile and lixiviated chrysotile had 80% viability, and on crocidolite 70% viability. Scanning electron microscopy showed that cells supported on lixiviated chrysotile are fully covered by the support, but crocidolite fibers adhere less, since they are stiffer. Fermentation experiments performed after 3 years storage showed that four from the five lixiviated chrysotile samples and one of the three crocidolite samples were active. In all cases, a delay time for the onset of fermentation was observed indicating a state of latency.     

Influence of chemical agents on interactions in dairy products: Effect of SDS on casein micelles

The addition of SDS during skim milk reconstitution is an original approach to study the effect of an ionic amphiphilic molecule on the milk system and particularly on the casein micelle component. SDS-induced changes in casein micelles were investigated by turbidimetry, rheology, scanning electron microscopy (SEM) and biochemical measurements (including soluble proteins analysis). This study shows that casein micelles were able to interact together to form micellar aggregates or milk gel without coagulating agents addition, when milk was reconstituted in the presence of SDS. This micellar aggregation, depending on the SDS concentration, is confirmed by SEM observations showing that the general aspect of casein micelles was affected by SDS treatment. Biochemical analysis indicated that SDS induced micellar casein dissociation. SDS-induced milk gel formation required a defined level of casein dissociation which could be also related to a particular micellar state.     

Structural ordering of casein micelles on silicon nitride micro-sieves during filtration

The paper reports on the structure and formation of casein micelle deposits on silicon nitride micro-sieves during the frontal filtration. The most frequent radius of the fractionated casein micelles we use is R=60 nm as detected by static light scattering (SLS) and atomic force microscopy (AFM). We estimate the size and size distribution of the casein micelles which pass through the micro-sieve during the filtration process. A sharpening of the size distribution at the beginning of the filtration process (t=40s) is followed by a broadening and a shift of the most frequent radii towards smaller sizes at later times (t=840 s). The size distribution of the micelles deposited on the micro-sieve during filtration is bimodal and consists of the largest and smallest micelles. At larger filtration times, we observe a shift of both deposited size classes towards smaller sizes. The atomic force micrographs of the reference sample reveal a tendency of the casein micelles to order in a hexagonal lattice when deposited on the micro-sieves by solution casting. The deposition of two size classes can be explained by a formation of a mixed hexagonal lattice with large micelles building up the basis lattice and smaller sizes filling octahedral and tetrahedral holes of the lattice. The accompanied compression with increasing thickness of the casein layer could result from preferential deposition of smaller sizes in the course of the filtration.     

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiresponsive Viscoelastic Vesicle Gels of Nonionic C12EO4 and Anionic AzoNa

Viscoelastic vesicle gels were prepared by mixing a nonionic surfactant, tetraethylene glycol monododecyl ether (C₁₂EO₄), and an anionic dye, sodium 4‐phenylazobenzoic acid (AzoNa). The gels, which were composed of multilamellar vesicles, were analyzed by cryogenic transmission electron microscopy (cryo‐TEM), freeze–fracture transmission electron microscopy (FF‐TEM), ²H NMR spectroscopy, and small‐angle X‐ray scattering (SAXS). The mechanism of vesicle‐gel formation is explained by the influence of anionic molecules on the bilayer bending modulus. Interestingly, the vesicle gels were observed to be sensitive to temperature, pH, and light. The viscoelastic vesicle gels respond to heat; they thin at lower temperatures and become thicker at higher temperatures. The vesicle gels are only stable from pH 7 to 11, and the gels become thinner outside of this range. UV light can also trigger a structural phase transition from micelles to multilamellar vesicle gels.     

Carrageenan Gels in Skim Milk: Formation and Rheological Properties

Phase behavior and mechanical properties of gels of three (kappa-, iota-, and lambda-) carrageenans in skim milk are thoroughly studied. The measurements were performed with the aid of a dynamic rheology in different regimes including determination of frequency dependences of rheological parameters, creep, and critical strain and stress at which the gelled systems are destroyed. Temperature dependences of the rheological parameters were also measured. A set of obtained data allowed to quantitatively characterize the mechanical properties of milk gels and compare the gel-forming properties of various carrageenans. In addition to gels of individual polysaccharides, their mixed gels were studied for the first time. It is shown that kappa-carrageenan formed the most stiff and brittle gels. Its mixing with iota- or lambda-carrageenans made gels softer. This effect is observed when the degree of substitution of kappa-carrageenan was higher than 50% that indicates its prevalent role in the mixed systems. The gelation is explained by the formation of crosslinked structure where the nodes are the casein micelles connected by polysaccharide chains. Their binding occurs due to electrostatic interactions between the sulfo groups of carrageenans and amino groups of kappa-casein. In the case of kappa- or iota-carrageenans, network structure is additionally stabilized due to crosslinking of segments of their molecular chains by the double helices at the gaps between casein micelles.     

Hierarchical networks of casein proteins: an elasticity study based on atomic force microscopy

2D- and 3D-atomic force microscopy (AFM) experiments were performed on single casein micelles (CM) in native state, submerged in liquid, using a home-built AFM instrument. The micelles were immobilized via carbodiimide chemistry to a self-assembled monolayer supported on gold-coated slides. Off-line data analysis allowed the extraction of both surface topography and elastic properties. Relative Young moduli (E*) were derived from force-vs-indentation curves, using the Hertz theory. The obtained E* values were found to increase with CM diameter, following a straight line dependence. The data showed that temperature, via its influence on both the protein-protein interactions and the composition of the micelle, has a clear effect on the mechanical properties of the CMs: higher temperatures and lower serum casein concentrations result in stiffer micelles. For pH < or = 5.6, effecting calcium phosphate release from the micelles by decreasing the pH does not have a large effect on CM stiffness. On decrease of the pH below 5.0, particulate gels and multilayers were obtained. Their measured elasticity (expressed by an equivalent G'AFM) agrees remarkably well with the storage moduli as measured with a conventional rheometer. Compared to single micelles, gels from nonheated CM suspensions are about 3 orders of magnitude softer. The "softness" of these gels (measured under compression or shear) therefore must come from the microscopic and/or mesoscopic links rather than the micelles themselves.     

Controlling sol-gel properties enhancing entrapped membrane protein activity through doping additives

Chlorophyllase, a membrane protein, was entrapped in tetramethoxysilane (TMOS)—derived sol-gel and activity examined through a modification of sol-gel properties with doping additives. Polyvinyl alcohol or polyethyleneglycol as uncharged polymeric additives reduced the activity of entrapped chlorophyllase by 60 and 70% respectively, explained by restricted accessibility of chlorophyllase, as revealed by nitrogen desorption experiments. Entrapped chlorophyllase demonstrated 10% activity yield in polyethylenimine-doped gel relative to a polyethylenimine-free control, explained by electrostatic interaction between positively charged polyethylenimine and negatively charged chlorophyllase, considering that polyethylenimine led to a gel with a slightly reduced specific surface area and pore volume, but 20% larger pore size. When chlorophyllase was introduced into sol-gel together with various amounts of glycerol, it demonstrated only slightly lower activity in those gels, even though sol-gel demonstrated decreased specific surface area and pore volume with increasing amount of glycerol. Lipase, as additive, reduced activity of entrapped chlorophyllase, explained by its effect on gel formation, and thus gelation time and gel properties, whereas activity of sol-gel entrapped chlorophyllase associated with monogalactosyl diglyceride (MGDG), was increased by 40%. This study provides an in-depth understanding of the change in sol-gel properties as affected by a wide variety of additives, and the consequent affect on the activity of the membrane protein chlorophyllase.     

Characterization of the pore structure of aqueous three-dimensional polyacrylamide gels with a novel cross-linker

The properties of polyacrylamide hydrogels synthesized with a novel hexafunctional (three double bonds) cross-linker, hexahydro-1,3,5-triacryloyl-s-triazine (1a), was evaluated and compared to the currently used tetrafunctional (two double bonds) cross-linker N,N-methylenebisacrylamide (Bis). A variety of characterization techniques that require very little sample preparation and data handling were chosen and include polymerization temperature profiles and conversions, water swelling, differential scanning calorimetry (DSC), polyacrylamide gel electrophoresis (PAGE), Gradiflow electrophoretic separation process and scanning electron microscopy (SEM). The alternative use of 1a compared to Bis results in polyacrylamide gels with larger pore sizes and a broad pore size distribution.     

Hydrophilic lecithins protect milk proteins against heat-induced aggregation

In the present study, the heat-induced interaction between whey proteins and casein micelles was studied. To that end, the particle size distribution of 5.5% (w/w) casein micellar dispersions was determined by photon correlation spectroscopy as a function of both the whey protein concentration and heating time at 80 °C. The results clearly indicated that heat-induced aggregation of the casein micelles only occurred in the presence of whey proteins.

In an effort to overcome the heat-induced interactions between whey proteins and casein micelles, the influence of different soybean lecithins was investigated. Comparing native to hydrolysed, as well as hydroxylated soybean lecithin, it was observed that the heat-stabilising effect of the lecithins was directly related to their hydrophilicity: whereas native soybean lecithin had hardly any beneficial effect, highly hydrolysed as well as hydroxylated soybean lecithin largely prevented heat-induced casein micelle aggregation in the presence of whey proteins.

From experimental observations on the heat-induced decrease of whey protein solubility both in the absence and presence of hydrolysed lecithin, it was deduced that the latter may stabilise the exposed hydrophobic surface sites of heat-denatured whey proteins. Dynamic surface tension measurements indicated that the heat-stabilising properties of lecithins were mainly determined by their critical aggregation concentration.     

Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering

Casein micelles are colloidal protein-calcium-transport complexes whose structure has not been unequivocally elucidated. This study used small-angle x-ray scattering (SAXS) and ultrasmall angle x-ray scattering (USAXS) as well as cryo transmission electron microscopy (cryo-TEM) to provide fine structural details on their structure. Cryo-TEM observations of native casein micelles fractionated by differential centrifugation showed that colloidal calcium phosphate appeared as nanoclusters with a diameter of about 2.5 nm. They were uniformly distributed in a homogeneous tangled web of caseins and were primarily responsible for the intensity distribution in the SAXS profiles at the highest q vectors corresponding to the internal structure of the casein micelles. A specific demineralization of casein micelles by decreasing the pH from 6.7 to 5.2 resulted in a reduced granular aspect of the micelles observed by cryo-TEM and the existence of a characteristic point of inflection in SAXS profiles. This supports the hypothesis that the smaller substructures detected by SAXS are colloidal calcium phosphate nanoclusters rather than putative submicelles.     

Changes in commercial wood charcoals by thermal treatments

As a continuation of a recent study of commercial wood charcoals as far as their potential production of carbon adsorbents is concerned, we have studied the influence of the final heating temperature (T) as carbonization variable in the range 250–1000 °C on the yield and on the characteristics of granular chars prepared from two very different charcoals: a holm-oak charcoal manufactured by partial combustion in a charcoal kiln and an eucalyptus charcoal industrially manufactured in a continuous furnace. Our study also includes the changes produced in both charcoals heated at 250 °C in air for 24 h, and their influences on the adsorption of water vapour at 25 °C. The samples were characterized by thermogravimetry, chemical analyses, Fourier transform infrared spectroscopy, densimetric measurements and mercury porosimetry. T affects the char yield, the chemical composition and the porosity of each char series differently. In particular, the total open pore volumes (to helium) of the starting charcoals, 0.475 and 1.044 cm³ g⁻¹, increase to 0.872 and 1.293 cm³ g⁻¹ heating up to 1000 and 500 °C, respectively. The changes by carbonization are mainly due to devolatilization; moreover, a moderate structural shrinkage occurs heating the eucalyptus charcoal at T > 500 °C. Concerning the air treatments, the yields do not present a significant difference; carbonyl groups are formed and the resulting pore structures depend on the starting charcoals. Water adsorption is larger for the eucalyptus carbons (approximately type V isotherms) than for the holm-oak carbons (type II isotherms).     

Protein Loading Capacity and Textural Properties of Column Packings in Reversed-Phase HPLC

Abstract

The relationship between the textural properties (pore size, pore volume and surface area) of reversed-phase silica gel packings for HPLC and the dynamic loading capacity of large biomolecules was studied by using silica gels manufactured by similar processes. Several silica gels whose unbonded pore diameters range from 100 to 250 A and whose pore volumes range from 1.0 to 1.4 ml/g have been prepared and characterized. The bonded phase is monomeric C18. The textural properties of the bonded silica gels are also presented and related to the properties of the unbonded silica gels.

Chromatographic evaluation with typical proteins in an underload-to-overload condition was performed in order to relate the influence of textural properties of silica gel to loading capacity and resolution. The packings with larger pore size and pore volume produced better column performance and higher loading of proteins.     

Analysis by DSC of the drying and sintering processes of alkoxide-derived SiO2–ZrO2 gels

The drying and sintering processes of SiO₂–ZrO₂ alkoxide-derived gels have been studied by means of DSC technique. In the drying process, most part of water and alcohols are removed from the gels. For the SiO₂ gel such elimination occurs at the end of the drying process, however for the ZrO₂ gel this elimination occurs during the whole drying time. An intermediate behavior is observed for the binary system SiO₂–ZrO₂ gels. In the sintering process, the DSC technique allows to determine the elimination of water and alcohols retained within the structure (open or close pores) and the well-known hydroxyl condensation of silica gel between 700° and 800°C is also observed. The ZrO₂ gel shows the final hydroxyl condensation at the heating temperature of 600°C. For the binary SiO₂–ZrO₂ gels, the hydroxyl condensation has been associated to the activation energy needed for the dissociation of silica hydroxyls. This energy decreases with the ZrO₂ concentration in the gel resulting in a sintering treatment of 500°C leading to the entire hydroxyl condensation for the gel with 75% ZrO₂–25% SiO₂.

By studying the temperature of the DSC peaks, it is possible to know the temperature at which most part of water and alcohols are leaving the gel, and these results can be used in order to select the corresponding drying or sintering schedules for obtaining a well-fabricated material.     

Formation of cellulose acetate membranes using a supercritical fluid assisted process

Microporous cellulose acetate membranes have been prepared from polymer–acetone solutions using a supercritical fluid phase inversion process in which CO₂ acts as the non-solvent. Series of experiments were performed at various polymer concentrations, temperatures and pressures. The structure of the resulting membranes was analysed using scanning electron microscopy. We operated with polymer concentrations ranging between 5 and 40% (w/w) in acetone obtaining different pore dimensions and membrane structures. Increasing the percentage of polymer in the solution, the structure of the membranes changed from beads-like structure to cellular structure. Polymer concentration also influenced the mean diameter of the pores that ranged from 2 to 50 μm for polymer concentrations from 40 to 5% (w/w). We also tested membrane formation pressures between 100 and 200 bar and at temperature between 45 and 65 °C. Pressure influences the change in membrane structure from cellular to beads-like, whereas temperature has a minor influence on pore size: both the effects can be partially related to CO₂ density. Cellulose acetate membrane formation mechanisms have also been discussed.